Enzymically active subcomplexes were purified from bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I) by sucrose-gradient centrifugation in the presence of detergents. These subcomplexes, named I lambda, IS, and I lambda S, catalyse ferricyanide and ubiquinone-1 (Q-1) reduction by NADH at similar rates to complex I, but do not catalyse the reduction of decylubiquinone. In addition, the Q-1 reductase activity of all the subcomplexes is insensitive to rotenone. Chemical and EPR analyses of the subcomplexes show that FMN and all the Fe-S clusters of complex I are present, but that the line shape of cluster 2 is modified. The smallest subcomplex, I lambda S, contains only approximately 13 subunits, as compared to approximately 22 in the previously described subcomplex I alpha [Finel, M., Skehel, J. M., Albracht, S. J. P., Fearnley, I. M. & Walker, J. E. (1992) Biochemistry 31, 11425-11434], but it retains the 75-, 51-, 49-, 30-, 24-, 23- (TYKY) and 20-kDa (PSST) subunits, which are suggested to form a functional core that comprises the EPR-detectable Fe-S clusters 1-4, and FMN. The structural and functional implications of such an arrangement are discussed.